Multilocus functional characterization of indigenous and exotic inbreds for dgat1-2, fatb, ge2 and wri1a genes affecting kernel oil and fatty acid profile in maize.

Demand for maize oil is progressively increasing due to its diverse industrial applications, aside from its primary role in human nutrition and animal feed. Oil content and composition are two crucial determinants of maize oil in the international market. As kernel oil in maize is a complex quantitative trait, improving this trait presents a challenge for plant breeders and biotechnologists. Here, we characterized a set of 292 diverse maize inbreds of both indigenous and exotic origin by exploiting functional polymorphism of the dgat1-2, fatb, ge2, and wri1a genes governing kernel oil in maize. Genotyping using gene-based functional markers revealed a lower frequencies of dgat1-2 (0.15) and fatb (0.12) mutant alleles and a higher frequencies of wild-type alleles (Dgat1-2: 0.85; fatB: 0.88). The favorable wri1a allele was conserved across genotypes, while its wild-type allele (WRI1a) was not detected. In contrast, none of the genotypes possessed the ge2 favorable allele. The frequency of favorable alleles of both dgat1-2 and fatb decreased to 0.03 when considered together. Furthermore, pairwise protein-protein interactions among target gene products were conducted to understand the effect of one protein on another and their responses to kernel oil through functional enrichments. Thus, the identified maize genotypes with dgat1-2, fatb, and wri1a favourable alleles, along with insights gained through the protein-protein association network, serve as prominent and unique genetic resources for high-oil maize breeding programs. This is the first comprehensive report on the functional characterization of diverse genotypes at the molecular and protein levels.

Pollen staining is a rapid and cost-effective alternative to marker-assisted selection for recessive waxy1 gene governing high amylopectin in maize.

Waxy maize is popular for food-, feed- and industrial usage. It possesses a recessive waxy1 (wx1) gene that enhances amylopectin to ~ 95-100%, compared to ~ 70-75% in traditional maize. Marker-assisted selection (MAS) is a preferred approach to converting normal maize into a waxy version. However, it requires specialized expertise, a well-equipped laboratory, and high cost. Here, pollen staining was used as an alternative approach to MAS. BC1F1, BC1F2 and BC2F2 populations in seven genetic backgrounds segregating for the wx1 gene were used. Pollens treated with iodine-potassium iodide showed that wild types (Wx1Wx1) were dark purple, while waxy pollens (wx1wx1) exhibited red colour. Heterozygotes (Wx1wx1) showed a mix of both dark purple and red colour. Staining of endosperm flour also confirmed the same findings. Wx1-based genotyping using phi022 and wx2507F/RG confirmed the same genotypic status. The average amylopectin among genotypes having red coloured pollens was 97.6%, while it was 72.5% among dark purple. Heterozygotes with both dark purple and red pollens had 85.2% amylopectin. Pollen staining showed complete agreement with the genotyping as well as amylopectin contents. Pollen staining saved 81% cost, and 54% time compared to MAS. This is the first report on the utilization of pollen staining for selecting the wx1 allele in segregating populations used for the development of waxy maize hybrids. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01240-1.

Allelic Variation in Zmfatb Gene Defines Variability for Fatty Acids Composition Among Diverse Maize Genotypes.

Edible oil with lower saturated fatty acids is desired for perceived quality and health benefits to humans and livestock. fatb gene encoding acyl-ACP thioesterase is a key player in the conversion of palmitic acid to oleic acid, thereby modifying the ratio of saturated to unsaturated fatty acids in maize kernels. The present investigation characterised the full-length sequence of the Zmfatb gene (4.63 kb) in two mutants (Zmfatb) and eight wild-types (ZmfatB) inbreds to study allelic variation, gene-based diversity, phylogenetic-relationship, protein-modelling, and molecular-docking to identify novel candidates for modification of fatty acid profile. Sequence alignment revealed wide genomic variability for Zmfatb among the inbreds; identified five novel SNPs and two InDels that clearly differentiated the wild-type and mutant genotypes. Gene-based diversity using 11-InDel markers categorised 48-diverse maize-inbreds into two-clusters. The majority of mutant and wild-type inbreds were grouped in separate clusters and led to the generation of 41 haplotypes. Genetic relationship of maize fatb gene with orthologues among 40 accessions of 12 oilseed-crops using both nucleotide and protein sequence clustered maize, soybean, sunflower, opium-poppy, Citrulus lanata, quinoa, and prunus species into one cluster; and brassica, camelina, and arabidopsis into the different cluster. The clustering pattern revealed that the plant oil with higher unsaturated fatty acids, particularly oleic, linoleic, and linolenic acids grouped together in one cluster and higher proportions of other fractions like arachidic, eicosenoic, and erucic acids grouped in another cluster. Physico-chemical properties highlighted more similarity between maize and 29 orthologue proteins, but orthologues were found to have better thermostability. Homology models have been developed for maize mutant and wild-type inbreds using Umbellularia californica (PDB ID: 5x04) as a template. Predicted protein models possessed optimum confidence-score and RMSD values and validated stability via., Ramachandran plots. Molecular docking indicated most of the interactions of protein-ligand were having similar binding-affinity due to the broader specificity of fatty acyl-ACP thioesterases and the presence of conserved-domains across crops. This is the first report on the comprehensive molecular characterisation of the fatb gene in maize and various orthologues. The information generated here provided new insights into the genetic diversity of fatb gene which can be utilised for the enhanced nutritive value of oil in the breeding programme.

Development of multinutrient-rich biofortified sweet corn hybrids through genomics-assisted selection of shrunken2, opaque2, lcyE and crtRB1 genes.

Sweet corn has gained worldwide popularity. Traditional sweet corn possesses low concentration of essential nutrients such as lysine (0.15-0.25%), tryptophan (0.03-0.04%) and provitamin-A (proA 3-4 ppm), and deficiency leads to serious health problems in humans. Here, stacking of shrunken2 (sh2), opaque2 (o2), lycopene epsilon cyclase (lcyE) and ß-carotene hydroxylase (crtRB1) genes  were undertaken in the parents of four hybrids viz., APQH1, APHQ4, APHQ5 and APHQ7 using marker-assisted backcross breeding (MABB). Gene-linked markers (umc2276 and umc1320) for sh2, while gene-based markers for o2 (umc1066 and phi057), lcyE (5'TE-InDel) and crtRB1 (3'TE-InDel), were used for genotyping in BC1F1, BC2F1 and BC2F2. Selected backcross progenies showed high recovery of recurrent parent genome (92.4-97.7%). The reconstituted sweet corn hybrids possessed significantly high lysine (0.390%), tryptophan (0.082%) and proA (21.14 ppm), coupled with high kernel sweetness (brix 18.96%). The improved sweet corn hybrids had high cob yield (12.22-15.33 t/ha) across three environments. These newly developed biofortified sweet corn hybrids possess great significance in providing balanced nutrition. This is the first report of combining sh2, o2, lcyE and crtRB1 genes for enrichment of sweet corn hybrids with multiple essential nutrients.

Molecular Breeding for Nutritionally Enriched Maize: Status and Prospects.

Maize is a major source of food security and economic development in sub-Saharan Africa (SSA), Latin America, and the Caribbean, and is among the top three cereal crops in Asia. Yet, maize is deficient in certain essential amino acids, vitamins, and minerals. Biofortified maize cultivars enriched with essential minerals and vitamins could be particularly impactful in rural areas with limited access to diversified diet, dietary supplements, and fortified foods. Significant progress has been made in developing, testing, and deploying maize cultivars biofortified with quality protein maize (QPM), provitamin A, and kernel zinc. In this review, we outline the status and prospects of developing nutritionally enriched maize by successfully harnessing conventional and molecular marker-assisted breeding, highlighting the need for intensification of efforts to create greater impacts on malnutrition in maize-consuming populations, especially in the low- and middle-income countries. Molecular marker-assisted selection methods are particularly useful for improving nutritional traits since conventional breeding methods are relatively constrained by the cost and throughput of nutritional trait phenotyping.

Marker-assisted pyramiding of opaque2 and novel opaque16 genes for further enrichment of lysine and tryptophan in sub-tropical maize.

The improvement of protein quality in maize so far has been based on recessive opaque2 (o2) mutant that along with endosperm-modifiers led to development of quality protein maize (QPM). Recent discovery of nutritional benefits of recessive opaque16 (o16) mutant was of immense significance for further improvement of protein quality. In the present study, o16 was introgressed into o2-based parental inbreds (HKI161, HKI193-1, HKI193-2 and HKI163) of four commercial QPM hybrids (HQPM-1, HQPM-4, HQPM-5 and HQPM-7) released in India, using marker-assisted backcross breeding. Background selection led to high recovery of recurrent parent genome (RPG) to maximum of 95%, and introgressed progenies showed considerable phenotypic resemblance for plant-, ear- and grain- characteristics to their respective recurrent parents. Selection of markers for o2 and o16 led to development of pyramided lines (o2o2/o16o16) that possessed as high as 76% and 91% more lysine and tryptophan over the recurrent parents, respectively. Reconstituted hybrids showed an average enhancement of 49% and 60% in lysine and tryptophan over the original hybrids, with highest enhancement amounting 64% and 86%, respectively. This is first report of enhancement of both lysine and tryptophan by o16 in maize genotypes adaptable to sub-tropics. Moderate variation in lysine and tryptophan was also observed in pyramided lines. Multi-location evaluation of reconstituted hybrids revealed similar grain yield and attributing traits to their original versions. This study signified the role of o16 as supplementary to o2 for nutritional quality enhancement in maize, and improved elite inbreds and hybrids developed here hold great significance in maize biofortification programme.

ß-Carotene bioaccessibility from biofortified maize (Zea mays) is related to its density and is negatively influenced by lutein and zeaxanthin.

Biofortification of maize with provitamin A (pro-VA) carotenoids has been successful, but the bioavailability of carotenoids needs to be explored. In the present study, we investigated the carotenoid content, micellarization and intestinal cell uptake of carotenoids from 10 maize hybrids [normal maize, quality protein maize (QPM), pro-VA carotenoid and double biofortified QPM + pro-VA maize hybrids] using a simulated in vitro digestion/Caco-2 cell model. The pro-VA carotenoid content of biofortified maize hybrids is 2-10 fold higher compared to that of normal maize. Furthermore, the ratio of non-pro-VA carotenoids lutein (LUT) plus zeaxanthin (ZEA) to the sum of pro-VA carotenoids ß-cryptoxanthin (BCX), α-carotene (AC) and ß-carotene (BC) in biofortified maize was much lower compared to that of normal maize. The consumption of 200 g day-1 of biofortified Pusa-PV-16-3 (BC = 808.4 µg per 100 g; AC = 839.3 µg per 100 g; BCX = 59 µg per 100 g) and Pusa-APQH8 (BC = 345.9 µg per 100 g; AC = 1739 µg per 100 g; BCX = 644.2 µg per 100 g) maize would contribute to 52 and 64% of RDAs for adult Indian men, respectively, after adjusting for cooking losses and conversion factors. The mean efficiency of micellarization of LUT (62.2% ± 5.3), ZEA (65% ± 4.7), and BCX (54% ± 9.5) exceeded that of AC (43% ± 8.9) and BC (49.8% ± 7.8) from all the maize hybrids. Furthermore, the micellarization and uptake in Caco-2 cells during a 4 h incubation period showed high correlation (P < 0.05) with the concentration of carotenoids in the maize digesta and micellar fraction, respectively. However, the LUT + ZEA content in the maize digesta and micellar fraction was inversely (p < 0.05) related to the BC micellarization and intestinal cell uptake, respectively. These results together suggest that the enrichment of pro-VA carotenoids together with decreasing the oxygenated carotenoid metabolites such as LUT and ZEA will further improve the bioavailability of BC from maize hybrids.

Nutritional security through crop biofortification in India: Status & future prospects.

Malnutrition has emerged as one of the most serious health issues worldwide. The consumption of unbalanced diet poor in nutritional quality causes malnutrition which is more prevalent in the underdeveloped and developing countries. Deficiency of proteins, essential amino acids, vitamins and minerals leads to poor health and increased susceptibility to various diseases, which in turn lead to significant loss in Gross Domestic Product and affect the socio-economic structure of the country. Although various avenues such as dietary-diversification, food-fortification and medical-supplementation are available, biofortification of crop varieties is considered as the most sustainable and cost-effective approach where the nutrients reach the target people in natural form. Here, we have discussed the present status on the development of biofortified crop varieties for various nutritional and antinutritional factors. Ongoing programmes of the Indian Council of Agricultural Research on the improvement of nutritional traits in different crops have been presented. Challenges and future prospects of crop biofortification in India have also been discussed. The newly developed biofortified crop varieties besides serving as an important source for livelihood to poor people assume great significance in nutritional security.